ABSTRACT/SUMMARY (Project 2) Tumor cell intrinsic stress including oncogene activation, as well as extrinsic stresses, such as low oxygen/nutrient availability, elicit perturbations in the endoplasmic reticulum (ER). Moreover, oncogenically transformed cells face increased burden placed by augmented biosynthetic pathways and rewired metabolism to meet the demands imposed by rapid proliferation. Adaptation to the ensuing stress and re-establishment of cellular homeostasis is achieved via activation of a coordinated signal transduction program termed the Integrated Stress Response (ISR). During the previous funded period, we demonstrated that increased rates of protein synthesis elicited by oncogenic MYC, activate the PERK/GCN2?eIF2? arm of the ISR, thereby supporting MYC-induced cell transformation. In preliminary unpublished studies, we have accumulated strong evidence supporting an essential role for the ISR effector and target of eIF2? ATF4, in transformation and tumorigenesis, particularly in tumors with activated MYC. However, how ATF4 elicits differential responses to various stresses in the context of MYC-dependent transformation is a critical question that remains unanswered. We will test the hypothesis that activation of ATF4 by the ISR plays a critical role in MYC- induced transformation and tumor progression by promoting metabolic and translational adaptation in coordination with MYC by focusing on three specific Aims. In Aim 1 we will identify critical nodes in cellular metabolism and translational regulation which are coordinately regulated by both ATF4 and c-MYC. Specifically, we will delineate the mechanism of Glut-1 and eIF4E transcriptional activation by ATF4 and determine functional requirements of GLUT1 and eIF4E in regulating glycolysis, translation and survival during MYC-dependent transformation in vitro and in vivo. Under Aim 2, we will delineate the mechanism of co- regulation of transcriptional targets between ATF4 and MYC by ChIP-seq analysis in lymphoma, colorectal (CRC) and prostate (PCa) cancer cells expressing inducible forms of MYC and analyze coordinately regulated genes. We will then determine the effects of knockout/knockdown of the identified co-regulated genes and newly identified targets in MYC-dependent proliferation, apoptosis and tumor growth. Finally, under Aim 3, we will determine the role of ATF4 in MYC-dependent transformation and tumorigenesis in PCa and CRC tumors using a conditional knockout ATF4 model crossed with PTENfl/fl:MycTg, and mouse CRC models (orthotopic, syngeneic and spontaneous) as well as 3D CRC organoids. We will also work with Project 1 to analyze the regulation of ATF4-dependent downregulation of BMAL1 and Clock genes and their role in translation and lymphomagenesis. Finally, with Project 3, we will analyze the effects of ATF4 ablation on type I interferon pathway and viability/effector functions of tumor-infiltrating cytotoxic T lymphocytes. Completion of these aims will provide a better understanding of the critical role of ATF4 in MYC-dependent pro-tumorigenic processes and may uncover new targets for therapeutic intervention in these malignancies.